Expression Profile Analysis Of Plasma MRNA And LncRNA In Hypertensive Intracerebral Hemorrhage Based On High-throughput Sequencing

Background and purpose:In recent years,due to the progress of next-generation sequencing technology,people’s exploration of long non-coding RNA has deepened,and a number of studies have shown that lnc RNA expression profile is an important marker of disease or its developmental state^[1],such as a variety of human cancers^[2,3,4],T cell differentiation^[5]and development^[6],etc.,revealing that lnc RNAs and m RNAs can directly or indirectly participate in the regulation of gene expression of various diseases and progression,so they have broad prospects as biomarkers for disease diagnosis and treatment.Hypertensive intracerebral hemorrhage（HICH）is a common type of stroke with acute onset and poor prognosis,and usually the nerve damage is irreversible.Therefore,it is of great significance to explore biomolecular markers for early prediction,diagnosis and prognosis of HICH.In this study,the expression profiles of lnc RNA and m RNA of plasma exosomes in HICH patients were analyzed and compared by high-throughput sequencing technology,and the related signaling pathways of lnc RNAs and m RNA were further studied by GO/KEGG enrichment analysis,in order to better identify HICH-related molecules and their mechanisms in HICH,so as to more accurately evaluate the disease progression and prognosis of patients,and lay a foundation for effective prevention and treatment of HICH.Methods:In this study,hypertensive cerebral hemorrhage was confirmed by imaging methods,and plasma samples were collected from patients with HICH and healthy controls（CON）.Exosome extraction and purification kit isolation and extraction of exosomes,immunoblotting,NTA and transmission electron microscopy identification of extracted exosomes,RNA extraction by exosome RNA extraction kit,sodium microspectrophotometer to detect RNA concentration and purity,high-throughput sequencing to detect RNA expression profiles,differential analysis and identification of m RNAs and lnc RNAs expressed differently between hypertensive cerebral hemorrhage group and healthy control group,GO/KEGG enrichment analysis,Lnc RNAs with high differential expression multiples were selected for primer design,synthesis,reverse transcription and real-time PCR verification,and nucleic acid integrity and primer PCR amplification specificity were detected by agarose gel electrophoresis experiments.Results:1.In this study,a total of 6 patients with hypertensive cerebral hemorrhage were included in this study,including 5 males,1 female,and 6 healthy CONtrols,including 5males and 1 female,There was no significant difference in the proportion of gender,smoking and drinking between the hypertensive cerebral hemorrhage group and the CONtrol group.The mean age of the HICH patients and the CON group were 57 years old,and there was no significant difference.2.Exosome quality control:immunoblotting,nanoparticle size tracing analysis,transmission electron microscopy showed that effective exosomes were extracted.3.A total of 72002 lnc RNAs and 18793 m RNAs were obtained in this study.The main types of lnc RNAs are intergenic and exonic-antisense.Comparing healthy CONtrols（CON）,76 up-regulated lnc RNAs and 276 down-regulated lnc RNAs,9717up-regulated m RNAs,and 9076 down-regulated m RNAs were identified in the hich group.4.GO analysis showed that CIS target genes expressing lnc RNA differently in HICH were highly enriched in homophilic cell adhesion via plasma membrane adhesion molecules and phosphatidic acid binding compared with healthy CONtrols.The trans target gene of lnc RNAs showed high enrichment in nucleic acid phosphodiester bond hydrolysis,voltage-gated sodium channel complex and ubiquitin-like protein transferase activity;GO analysis of differentially expressed m RNAs showed high enrichment in positive regulation of inflammatory response,cytoplasmic side of plasma membrane,and RAGE receptor binding.5.KEGG pathway analysis showed that CIS target genes of lnc RNAs with different expression in the HICH group were enriched in 178 signaling pathways compared with CON,and had higher enrichment in the mineral absorption pathway.The trans target genes of lnc RNAs with different expression were enriched in 28 signaling pathways,and high enrichment in the Herpes simplex virus 1 infection pathway.The m RNAs with different expression were enriched in 144 signaling pathways,and were highly enriched in the PD-L1 expression and PD-1 checkpoint pathway in cancer pathways.6.Quantitative real-time PCR verification and agarose gel electrophoresis experiments showed that NONHSAG111409,LOC101927429 and AL078639.1 were highly expressed in HICH,and the specificity of PCR amplification of NONHSAG111409 primers was good.Conclusions:1.In this study,we combined exosomes with transcript high-throughput sequencing for the first time,and experimentally verified the differentially expressed lnc RNA and m RNA in plasma exosomes in the HICH group and the healthy control group,and obtained the differential expression profiles of lnc RNA and m RNA.2.The results showed that the differentially expressed lnc RNAs may participate in the regulation of cytoplasmic membrane molecule adhesion,hydrolysis of nucleic acid phosphodiester bonds,control voltage-gated sodium channel complexes,and regulate ubiquitin-like protein transferase activity through their CIS target genes and trans target genes,thereby participating in the regulation of HICH.3.The analysis of the results showed that differentially expressed m RNAs may participate in the progression of HICH disease through positive regulation of inflammatory response,binding to unilateral intrinsic components of the plasma membrane,and regulating the binding process of RAGF receptors.4.The results of this study speculate that differentially expressed lnc RNAs may play a key role in the interleukin 17 signaling pathway and the biosynthesis pathway of sphingolipids by acting on CIS and trans target genes,and that differentially expressed m RNAs may affect the inflammatory stress response and prognosis of HICH by participating in the T cell receptor signaling pathway,Th2 cell differentiation pathway and Th17 cell differentiation pathway.5.NONHSAG111409,LOC101927429 and AL078639.1 were highly expressed in HICH,and the PCR amplification of NONHSAG111409 primers was strong.